Omeprazole (OP), is a potent inhibitor of the H+, K+-ATPase pump in gastric parietal cells. OP oxidation is catalyzed by liver cytochrome P450s. Although the specific identity of the CYP enzyme responsible remains controversial, there is evidence that OP hydroxylation cosegregates with the polymorphism of S-mephenytoin 4'-hydrolxylation in man. We have shown previously that mephenytoin was metabolized principally by CYP2C19 in man. Present studies were undertaken to assess OP metabolism using human CYP2C recombinant enzymes expressed in yeast and in (AHH-1) (TK+/-) cells. Microsomes were incubated with 14C-OP and the supernatant was analyzed by HPLC. A single metabolite with the same HPLC retention time as that of 5-hydroxyomeprazole (5OH-OP) was the only metabolite formed by CYP2C19, CYP2C8, and CYP2C18. The rates of 5OH-OP formation by these enzymes were 13.4+1.r, 2.2+0.1, and 1.5+0.1 nmol/min/nmol P 450, respectively. In contrast, CYP2C9 and its mutants did not produce 5OH-OP. Incubation of OP with microsomes from (AHH-1) (TK+/-) cells expressing recombinant human CYP3A4 resulted in the formation of two principal metabolites. These were identified as 5OH-OP and omeprazole sulfone (OP-S). Inhibition studies using troleandomycin indicated that additional enzymes might be involved in omeprazole metabolism in human liver microsomes.